Novel pan-serotype control RNA for dengue virus typing through real-time reverse transcription-polymerase chain reaction

•A control plasmid for DENV serotyping through qRT-PCR was developed.•The qDENV Control plasmid allowed in vitro transcription of DENV-1 to -4 multi-target sequence.•The qDENV-Control RNA allowed to validate the qRT-PCR results corresponding to each DENV serotype.•The qDENV-Control RNA was valuable for absolute DENV quantification from unknown samples.•This control is relevant for DENV surveillance and elucidation of the disease dynamics. Dengue virus (DENV) is the causative agent of one of the most important febrile illnesses worldwide. Four DENV serotypes are responsible for a broad clinical spectrum of the disease. Positive controls are costly and required for the validation of molecular test results of DENV serotyping. In this study, we describe the in silico design of the qDENV-Control plasmid with the target sequences to oligonucleotides and probes widely used for DENV serotyping, and the subsequent production of qDENV Control RNA by T7-driven run-off in vitro transcription. The qDENV Control RNA was successfully used to validate the positive and negative DENV serotyping results, allowing its incorporation in routine in-house protocols for virologic surveillance. This Control RNA allowed the absolute quantification of viral RNA copies from unknown samples as required in several fundamental studies.